Project Summary Cochlear amplification is the process by which our auditory system amplifies and tunes responses to incoming sounds, bestowing us with our excellent sound level sensitivity, large dynamic range, and fine frequency discrimination. Auditory sensory cells have two processes hypothesized to contribute to cochlear amplification: somatic motility that occurs in the cell soma and active hair bundle mechanics that occurs in the apical stereocilia hair bundle. To assay the contribution of active hair bundle mechanics to cochlear amplification requires further understanding of the processes related to it. Hair cell mechanotransduction (MET), the process of converting sound stimuli into electrical signals in the hair bundle, is the driver of active hair bundle mechanics. MET adaptation is one key mechanism that is hypothesized to contribute to active hair bundle mechanics. Previous work in non-mammalian models show that adaptation is separated into fast and slow processes, both of which rely on the influx of calcium to drive the process. Data in the mammalian cochlea indicate that adaptation also consists of fast and slow components, but our work shows that the underlying biology driving the fast and slow processes in the cochlea is fundamentally different from what has been previously reported in non- mammalian hair cells. Thus, new investigations are needed to understand the molecular machinery responsible for both fast and slow adaptation, and their contributions to mammalian auditory processing. From new data about properties of cochlear MET, we hypothesize that tension is essential for adaptation mechanisms. In Aim 1 of this study, we will investigate the contribution of myosin motors to adaptation and hair bundle mechanics. We assay this using new, faster stimulation and high-speed imaging to monitor mechanical changes in the hair bundle coupled with hair cell electrophysiology and pharmacological manipulation. With numerous myosin motors known to be important for auditory function, in Aim 2 we will explore the contributions of specific myosin motors to adaptation and hair bundle mechanics using existing mouse models. For Aim 3, we developed a new mouse model using CRISPR/Cas9 technology to acutely inactivate myosin VIIa motor function, and we will assess the role of myosin VIIa in tension generation. The experiments in this proposal will further our understanding of the molecular mechanisms of mammalian cochlear adaptation and hair bundle mechanics to develop a new model of the mammalian auditory MET process. We are uniquely positioned to accomplish this with the new technologies that we have and continue to develop. Basic mechanistic knowledge of auditory MET will lead to experiments where we can interrogate the system in vivo to determine specific molecular contributions to cochlear amplification. Understanding cochlear amplification can lead to better prevention and/or restoration of hearing.